Inositol Pyrophosphates Determine Exocytotic Capacity

ABSTRACT

The invention provides reagents and methods for treating type II diabetes, as well as methods for identifying compounds for treating type II diabetes.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.12/199,388, filed Aug. 27, 2008 which claims priority to U.S.Provisional Patent Application Ser. No. 60/969,443 filed Aug. 31, 2007,incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Phosphoinositides, in both their water-soluble and lipid forms, have aprominent role in cellular signal-transduction events. Important eventsare the generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) andits regulation of intracellular Ca2+ homeostasis (1) and the3-phosphorylated inositol lipid products of phosphatidylinositol (PI3kinase) (2), with diverse roles in mitogenesis, apoptosis and vesicletrafficking. Phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2), themajor source of these two signalling systems, is not merely a precursorfor the above signal transduction pathways but plays in itselfsignificant roles in vesicle trafficking, exocytosis, cytoskeletalrearrangements and regulation of ion channels (3). In the last decadethere has also been a growing appreciation that highly phosphorylatedinositol polyphosphates, distant derivatives of the Ins(1,4,5)P3 secondmessenger, play a role in signal-transduction and cellular regulation(4-6). Perhaps the most exciting new vista that has opened concerns therole of diester derivatives of both inositol pentakis- andhexakisphosphates (InsP5 and InsP6). The pyrophosphate derivatives ofInsP6 diphosphoinositol pentakisphosphate, and bis-(dipbospho)inositoltetrakisphosphate are commonly referred to as ‘InsP7’ and ‘InsP8’. Theseinositol pyrophosphate derivatives rapidly turnover and are estimated tohave similar free energy of hydrolysis as ATP (4). A strikingconsequence of this high-energy phosphate group is the ability of InsP7to directly phosphorylate a subset of proteins in an ATP- andenzyme-independent manner (7). The variety of cellular responses,apparently controlled by these molecules (4,8) may be facilitated by thedifferential intracellular distribution of the kinases that make them(9). The concentrations of inositol pyrophosphates can be dynamicallyregulated during key cellular events, underscoring their importance forcell function. For example, InsP7 levels change during cell cycleprogression (10) and InsP7 regulates cyclin/CDK complexes (11) whereasInsP8 increases acutely in response to cellular stress (8). However,recent work has also demonstrated a role for InsP6 as an enzymaticco-factor and so by analogy, it is possible that even undernon-stimulatory conditions, InsP7 could be an important regulatorymolecule.

Phosphoinositides are also key regulators of the insulin secretingpancreatic β-cell (12). These cells are critical players in bloodglucose homeostasis and act by coupling increases in the concentrationof glucose and other circulatory or neuronal-derived regulators, to theexocytosis of insulin. The highly phosphorylated InsP6 is particularlyinteresting as it has been shown to activate voltage-dependent L-typeCa2+ channels (13), exocytosis (14,15) and dynamin-mediated endocytosis(16), all key processes in insulin secretion. A role for InsP7 in theβ-cell has not yet been determined. However, given the suggestedinvolvement of inositol pyrophosphates in vesicle trafficking (4), thecritical nature of such trafficking events for the process of insulinexocytosis and the high β-cell concentration of InsP6 (13), theimmediate precursor of InsP7, we postulated that inositol pyrophosphatesmay play a significant role in the β-cell. We now demonstrate a novelrole for InsP7 in the regulation of insulin exocytosis.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for treating typeII diabetes comprising administering to a patient with type II diabetesan effective amount of a therapeutic capable of increasing expression ofIP6K1 kinase.

In another aspect, the present invention provides methods forstimulating insulin exocytosis from pancreatic beta cells comprisingadministering to a patient in need thereof an effective amount of atherapeutic capable of increasing expression IP6K1 kinase.

In another aspect, the present invention provides methods for treatingtype II diabetes comprising administering to a patient with type IIdiabetes an effective amount of a therapeutic capable of increasingproduction of InsP₇.

In a further aspect, the present invention provides methods foridentifying a compound for treating type II diabetes comprising:

(a) contacting pancreatic beta cells with one or more test compounds;and

(b) determining expression level of IP6K1 kinase and/or levels of InsP7;

wherein an increase in the expression of IP6K1 kinase and/or an increasein InsP7 indicates that the compound is suitable for treating type IIdiabetes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. High basal levels of InsP7 are present in pancreatic β cells andIP6K's are expressed in these cells. (A) Comparison of [3H]-labeledInsP7 as a percentage of [3H]-labeled. InsP6 in primary pancreaticislets or insulin secreting MIN6m9 cells. Data are from 3 separateexperiments. (B) The islet data from (A) were transformed to take intoaccount the different β-cell composition of normal (60%) vs. ob/ob(90%), islets. (C). Total RNA was extracted from islets and MIN6m9 cellsand reverse transcribed. Relative expression of messenger RNA wasmeasured by quantitative Real time PCR using appropriate primers andprobes. Primers and probe for 18S rRNA (TaqMan™ Ribosomal RNA ControlReagents, Applied Biosystems) were used as endogenous control.

FIG. 2. Expression of IP6K's promote exocytosis in pancreatic β-cells.IP6K1 stimulates Ca2+-dependent exocytosis. (A) individual mouse β-cellswere transfected with EGFP (mock) or a combination of EGFP and either awild-type (IP6K1) or a kinase-dead (IP6K1-K/A) variant of IP6K andsubjected to a train of four 500-ms depolarizations using the perforatedpatch configuration. Increases in cell capacitance (ΔCm) were measuredat 3 mM glucose in the extracellular medium. (B) Histogram summarizingthe average increase in cell capacitance plotted against the individualdepolarizations as well as the total increase in cell capacitance at theend of the train in cells mock transfected or overexpressing eitherwild-type (IP6K1) or kinase-dead (IP6K1-K/A) IP6K. (c) Histogram showingintegrated Ca2+ current (QCa) plotted against the individualdepolarizations in cells mock transfected or overexpressing eitherwild-type or IP6K1-K/A. Values are from 8-12 experiments. *P<0.05. (D)Histogram summarizing the average total increase in cell capacitance atthe end of the train in mock transfected cells or cells overexpressingeither wild-type (IP6Kn) or kinase-dead (IP6Kn-K/A) type 1, 2 and 3kinases, respectively. Values are from 7-12 experiments, *P<0.05. (E)INS-1E cells were co-transfected in parallel with pCMV5-hGH and emptyvector (pcDNA3) (mock) or with pCMV5-hGH and either wild-type (IP6Kn)or, kinase-dead (IP6Kn-K/A), types 1, 2 and 3 kinases, respectively. hGHsecretion was measured in Krebs-Ringer bicarbonate HEPES buffer with 3mM glucose, hGH release is depicted as secreted hGH in percentage oftotal hGH. Values from 3 experiments (each in triplicate). *P<0.05.

FIG. 3. InsP7 dose-dependently promotes Ca2+-dependent exocytosis.Individual mouse β-cells were subjected to a train of four 500-msdepolarizations using the standard whole-cell patch configuration. (A)Exocytosis was observed under control conditions and in the presence of3 μM 5-InsP7 in the pipette-filling solution. 5-InsP7 was allowed todiffuse into the cell for 2 min before initiation of the experiment. (B)Histogram summarizing the average increases in cell capacitance plottedagainst the individual depolarizations as well as the total increases incell capacitance at the end of the train in the absence or presence of 3μM 5-InsP7 in the pipette-filling solution. (C) Histogram showingintegrated Ca2+ current (QCa) plotted against the individualdepolarizations in the absence or presence of 3 μM InsP7 in thepipette-filling solution. (D) Concentration dependence of stimulatoryaction of 5-InsP7 on exocytosis evoked by a single membranedepolarization from −70 mV to zero. The curve represents a least-squaresfit of the mean data points to the Hill equation, Values are from 5-7experiments. *P<0.05. (E) A comparison of several isomers of InsP7 at a10 μM concentration on exocytosis using the same protocols as in (A)above.

FIG. 4. RNA silencing of IP6K1 but not IP6K2 inhibits release ofgranules from the RRP. (A) Individual mouse β-cells were transfectedwith siRNA to IP6K1 (No. 1) at 25 nM or a negative control at the sameconcentration and subjected to a train of four 500-ms depolarizationsusing the perforated patch configuration. Increases in cell capacitance(ΔCm) were measured at 3 nM glucose in the extracellular medium. (B)Histogram summarizing the average increases in cell capacitance plottedagainst the individual depolarizations as well as the total increase incell capacitance at the end of the train in cells mock transfected oroverexpressing either siRNA to IP6K1 or negative control. (C) Effect ontotal capacitance increase following RNA silencing of IP6K1 and IP6K2.(D) Effect of 5-InsP7 on exocytosis in under control conditions and incells with reduced expression levels of IP6K1.

FIG. 5. Effect of 5-InsP7 on exocytosis is distinct from InsP6.Individual mouse β-cells were subjected to a train of four 500-msdepolarizations using the standard whole-cell patch configuration.Exocytosis was observed under control conditions and in the presence ofeither 3 μM 5-InsP7 or 10 μM InsP6 in the pipette-filling solution. Theinositol phosphates were allowed to diffuse into the cell for 2 minbefore initiation of the experiment.

FIG. 6. Screening siRNA's in MIN6m9 cells. Six siRNA's for each IP6Kwere screened for their ability to silence at 100 nM in MIN6m) cells.Two in each case IP6K1 (1 and 4) and IP6K2 (3 and 5) were then used inindividually or in combination to silence IP6K1 or IP6K2 respectively.This was compared to 2 negative controls. mRNA was extracted and theexpression of the genes quantified using Taqman™ RT-PCR. Data areaverages±SEM, n=3)

FIG. 7. RNA silencing of IP6K1 or IP6K2 lowers cellular InsP7 levels.MIN6m9 cells were transfected with selected siRNA for either negativecontrol or IP6K1 and 2. SiRNA's for IP6K1 (1 and 4) were added at 25 nMeach. Similar concentrations of the 2 siRNA's for IP6K2 (3 and 5) wereadded. This was controlled by addition of a 50 nM of a negative control.All 4 siRNA's were also applied simultaneously and controlled with 100nM negative control siRNA. Two hours after transfection with siRNAmedium was changed to a 50 μCi/ml. [3H]-inositol containing medium andcells were cultured for 48 h to 72 h. Cells were extracted and subjectedto HPLC. Data are expressed relative to total inositol lipid and aremeans from 3 separate experiments±SEM, n=3).

FIG. 8. Effect of IP6K1-siRNA on single L-type Ca2+ channel activity inMIN6m9 cells. MIN6m9 cells were transfected with selected siRNA foreither negative control 50 nM or siRNA's for IP6K1 (1 and 4) at 25 nMeach. (A) Examples of single Ca2+ channel currents recorded fromcell-attached patches on a control cell (negative control siRNAtransfection, left) and a cell subjected to IP6K1-siRNA (right). Bothpatches contain one L-type Ca2+ channel. (B) Single L-type Ca2+ channelcurrent parameters in control MIN6m9 cells (n=30) and those subjected toIP6K1-siRNA (n=30). There is no significant difference in channel numberper patch, open probability, mean closed time and mean open time betweencontrol MIN6m9 cells and those subjected to IP6K1-siRNA (P>0.05). Dataare presented as means±SEM. Statistical significance was evaluated byeither Mann-Whitney U test or unpaired Student's t-test.

DETAILED DESCRIPTION OF THE INVENTION

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog(Ambion, Austin, Tex.).

In one aspect, the present invention provides methods for treating typeII diabetes comprising administering to a subject with type II diabetesan amount effective to treat type II diabetes of a therapeutic capableof increasing InsP7 in pancreatic beta cells of the subject.

In a further aspect, the present invention provides methods for treatingtype II diabetes comprising administering to a subject with type IIdiabetes an amount effective to treat type II diabetes of a therapeuticcapable of increasing expression of IP6K1 kinase in pancreatic betacells of the subject.

As the inventors have demonstrated in the attached, the pancreaticβ-cell maintains high levels of InsP7. This pyrophosphate then serves asan essential player in the insulin secretory process by regulating thereadily releasable pool of insulin-containing granules and therebymaintaining the immediate exocytotic capacity of the β-cell. Theinventors further showed that endogenous InsP7 generated by IP6K1 isresponsible for the enhanced exocytotic capacity in pancreaticbeta-cells. Thus, therapeutics capable of increasing expression of IP6K1kinase can be used to treat type II diabetes by generating InsP7,resulting in increased exocytotic capacity in pancreatic beta cells.

In one embodiment, the therapeutic comprises a gene therapy vectordirecting expression of IP6K1 or active fragments thereof. (Proteinaccession information: Q 92551 (SEQ ID NO: 1); cDNA accessioninformation (Alternative splice variants) 1. NM_(—)153273.3 (SEQ ID NO:3), 2. NM_(—)001006115 (SEQ ID NO: 2)) comprises a gene therapy vectordirecting expression of IP6K1 or active fragments thereof. The genetherapy method comprises administration of a nucleic acid constructcapable of expressing IP6K1 or active fragments thereof in the subject,and preferably in pancreatic beta cells of the subject. In one example,the cDNA sequences may be operably linked with an insulin promoter(Leibiger, Mol. Cell. 1:933-938 (1998)). Such gene therapy and deliverytechniques are known in the art; see, for example, WO90/11092, which isherein incorporated by reference, or: M. I. Phillips (Ed.): Gene TherapyMethods. Methods in Enzymology, Vol. 346, Academic Press, San Diego2002. Thus, for example, cells from the subject may be engineered exvivo with a nucleic acid construct comprising a promoter operably linkedto the nucleic acid molecule corresponding to the molecule to beintroduced, with the engineered cells then being provided to the subjectto be treated. Such methods are well-known in the art. For example, seeBelidegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993);Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini,M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int.J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50:5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-10(1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); andZhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which areherein incorporated by reference. The cells which are engineered may be,for example, pancreatic beta cells.

The nucleic acid molecules may also be delivered as a naked nucleic acidmolecule. The term “naked” nucleic acid molecule refers to sequencesthat are free from any delivery vehicle that acts to assist, promote orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the nucleic acid molecules used in gene therapy canalso be delivered in liposome formulations and lipofectin formulationsand the like that can be prepared by methods well known to those skilledin the art. Such methods are described, for example, in U.S. Pat. Nos.5,593,972, 5,589,466, and 5,580,859, which are herein incorporated byreference.

The naked nucleic acid molecules are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art. The constructs may also be delivered with delivery vehiclessuch as viral sequences, viral particles, liposome formulations,lipofectin, precipitating agents, etc.

In another embodiment, the therapeutic comprises IP6K1 or activefragments thereof. The polypeptides can be administered via any suitabletechnique, including but not limited to delivery as a conjugate with atransduction domain, which are one or more amino acid sequence or anyother molecule that can carry an active domain across cell membranes.These domains can be linked to other polypeptides to direct movement ofthe linked polypeptide across cell membranes. (See, for example, Cell55: 1179-11188, 1988; Cell 55: 1189-1193, 1988; Proc Natl Acad Sci USA91: 664-668, 1994; Science 285: 1569-1572, 1999; J Biol Chem 276:3254-3261, 2001; and Cancer Res 61: 474-477, 2001)

In a further aspect, the present invention provides methods foridentifying a compound for treating type ii diabetes comprising:

(a) contacting pancreatic beta cells with one or more test compounds;and

(b) determining expression level of IP6K1 kinase and/or levels of InsP7;

wherein an increase in the expression of IP6K1 kinase and/or an increasein InsP7 indicates that the compound is suitable for treating type IIdiabetes.

As noted above, therapeutics capable of increasing expression of IP6K1kinase can be used to treat type II diabetes by generating InsP7,resulting in increased exocytotic capacity in pancreatic beta cells.Thus, compounds that can be used to increase expression of IP6K1 kinaseand/or InsP7 in pancreatic beta cells can be used to treat type IIdiabetes.

Determining expression levels of IP6K1 kinase and/or an increase inInsP7 in the pancreatic beta cells can be performed using any techniquein the art, including but not limited to those disclosed in the examplesthat follow.

As used herein, “basal glucose conditions” mean a glucose concentrationof between 1 and 6 mM glucose; in one embodiment, 3 mM glucose is used.As is understood by those of skill in the art, basal glucoseconcentration may vary between species. Basal glucose concentration canbe determined for any particular cell or tissue type by those conditionsthat do not induce changes in, for example, cytoplasmic free Ca2+concentration or insulin release.

As used herein, “pancreatic β cells” are any population of cells thatcontains pancreatic β islet cells. The cells can be obtained from anymammalian species, or may be present within the mammalian species whenthe assays are conducted in vivo. Such pancreatic β islet cellpopulations include the pancreas, isolated pancreatic islets ofLangerhans (“pancreatic islets”), isolated pancreatic β islet cells, andinsulin secreting cell lines. Methods for pancreatic isolation are wellknown in the art, and methods for isolating pancreatic islets, can befound, for example, in Cejvan et al., Diabetes 52:1176-1181 (2003);Zambre et al., Biochem. Pharmacol. 57:1159-1164 (1999), and Fagan etal., Surgery 124:254-259 (1998), and references cited therein. Insulinsecreting cell lines are available from the American Tissue CultureCollection (“ATCC”) (Rockville, Md.). In a further embodiment wherepancreatic β cells are used, they are obtained from ob/ob mice, whichcontain more than 95% β cells in their islets.

In order to derive optimal information on the ability of the one or moretest compounds to increase in the expression of IP6K1 kinase and/or anincrease in InsP7 in pancreatic beta cells, it is preferred to compareIP6K1 kinase and/or InsP7 levels ion experimental cells with levels fromcontrol cells. Such control cells can include one or more of thefollowing:

1. The same host cells, treated in the same way except not contactedwith the one or more test compounds;

2. The same host cells, treated in the same way except contacted withthe one or more test compounds at different time points (for analyzingtime-dependent effects); and

3. The same host cells, treated in the same way except contacted withdifferent concentrations of the one or more test compounds (foranalyzing concentration-dependent effects);

When the test compounds comprise polypeptide sequences, suchpolypeptides may be chemically synthesized or recombinantly expressed.Recombinant expression can be accomplished using standard methods in theart, as disclosed above. Such expression vectors can comprise bacterialor viral expression vectors, and such host cells can be prokaryotic oreukaryotic. Synthetic polypeptides, prepared using the well-knowntechniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (Nα-amino protected Nα-t-butyloxycarbonyi) amino acid resinwith standard deprotecting, neutralization, coupling and wash protocols,or standard base-labile Nα-amino protected 9-fluorenylmethoxycarbortyl(Fmoc) amino acids. Both Fmoc and Boc Nα-amino protected amino acids canbe obtained from Sigma, Cambridge Research Biochemical, or otherchemical companies familiar to those skilled in the art. In addition,the polypeptides can be synthesized with other Nα-protecting groups thatare familiar to those skilled in this art, Solid phase peptide synthesismay be accomplished by techniques familiar to those in the art andprovided, such as by using automated synthesizers.

When the test compounds comprise antibodies, such antibodies can bepolyclonal or monoclonal. The antibodies can be humanized, fully human,or murine forms of the antibodies. Such antibodies can be made bywell-known methods, such as described in Harlow and Lane, Antibodies; ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1988).

When the test compounds comprise nucleic acid sequences, such nucleicacids may be chemically synthesized or recombinantly expressed as well.Recombinant expression techniques are well known to those in the art(See, for example, Sambrook, et al., 1989, supra). The nucleic acids maybe DNA or RNA, and may be single stranded or double. Similarly, suchnucleic acids can be chemically or enzymatically synthesized by manualor automated reactions, using standard techniques in the art. Ifsynthesized chemically or by in vitro enzymatic synthesis, the nucleicacid may be purified prior to introduction into the cell. For example,the nucleic acids can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the nucleic acids may be used withno or a minimum of purification to avoid losses due to sampleprocessing.

When the test compounds comprise compounds other then polypeptides,antibodies, or nucleic acids, such compounds can be made by any of thevariety of methods in the art for conducting organic chemical synthesis.

Test compounds identified as increasing the expression of IP6K1 kinaseand/or an increase in InsP7 in the pancreatic beta cells can be furtherassessed for use as a candidate compound for treating type II diabetesusing any further technique, including but not limited to contactingpancreatic beta cells with the test compounds and measuring insulinrelease induced by the test compounds, and/or by measuring resultingpancreatic beta cell capacitance induced by the test compounds; thosecompounds that increase insulin release and/or capacitance (which is ameasure of insulin exocytosis as described below) compared to controlmay be of particular value as candidate compounds for treating type IIdiabetes. In a further embodiment, measuring capacitance is performed asdescribed below, and those test compounds that elicit an exocytoticresponse at the first depolarization are considered good candidatecompounds for treating type II diabetes.

EXAMPLES Materials and Methods

Reagents and constructs. 5-Diphosphoinositol pentakisphosphate (InsP7)was synthesized as described previously (25). The ORF for IP6K1, IP6K2and IP6K3, were obtained by digestion using SalI-NotI pCMV-IP6K1,pCMV-IP6K2 (26) and by digestion using San pGST-IP6K3 (27). The purifiedORF were subcloned in the eukaryotic expression vector pCMV-Myc(Clontech). Kinase-dead versions were prepared as follows. Previousstudies have identified a lysine InsP3KA that is critical for catalyticactivity (28). In mouse IP6K1, human IP6K2 and human IP6K3 this lysineoccurs at position 226, 222 and 217, respectively. For IP6K1 we mutatedlysine 226 to alanine using the following oligo: K26A,5′-GTGTGCTGGACTTGGCCATGGGTACCCG-3′ (SEQ ID NO: 4) and complement. ForIP6K2 we mutated lysine 222 to alanine using the following oligo: K222A,5′-GTCCTTGACCTCGCGATGGGCACACGA-3′ (SEQ ID NO: 5) and complement. ForIP6K3 we mutated lysine 217 to alanine using the following oligo: K217A,5′-CCCEGTGTCCTGGATCTGGCCATGGGGACCCGGCAGCAC-3′ (SEQ ID NO: 6) andcomplement.

Constructs were tested in INS-1E cells to establish their efficacy.IP6K1-3 and their respective catalytically inactive forms weretransfected into INS-1E cells (protocols below). All constructs wereexpressed at similar level, as judged by western blotting. Moreover,IP6K1-3 wt, but not their catalytically inactive forms (K/A) increasedcellular InsP7 up to 6-fold.

RNAi's were Obtained from Ambion Inc (Austin, Tex.) and the followingRNAi ID's were used to silence IP6K's. RNAi's to IP6K1 (1, siRNAiID=188560) and (4, siRNAi ID=71758). RNAi's for IP6K2 (3, siRNAID=287702) and (5, siRNA ID=292211). Non-targeting controls (1, siRNA ID4611) and (2, siRNA ID=4613) were used as negative controls. ThesesiRNA's were also supplied by Ambion with Cy3 fluorescent tags and usedin the primary mouse beta cell experiments.

RNA Extraction and Real Time-PCR.

Total RNAs were extracted from cells using the RNeasy™ Micro Kit (QiagenInc, Valencia, Calif.). The RNAs were digested with DNase I for 1 hourat 37° C. (Fermentas, St. Leon Rot, Germany) and then re-purified withRNeasy™ Micro Kit (Qiagen Inc). The Applied Biosystem MultiScribe™Reverse Transcriptase kit was used to reverse transcribe 1 μg ofpurified RNA according to manufacture's instructions. 3.94 μl of theresulting cDNAs from the reverse transcriptase reaction were diluted in10.06 μl sterile water and 1.25 μl aliquots of each sample were testedin triplicate for each different quantitative PCR reaction. Relativeexpression of messenger RNA was measured by quantitative RT-PCR (withTaqMan Gene Expression Assays products on an ABI PRISM™ 7700 SequenceDetection System, Applied Biosystems, Foster City, Calif.). For theanalysis, the following TaqMan™ assays (Applied Biosystems) were used:for inositol hexaphosphate kinase 1, for IP6K2: inositol hexaphosphatekinase 2 and for IP6K3: inositol hexaphosphate kinase 3. Primers andprobe for 18S rRNA (TaqMan™ Ribosomal RNA Control Reagents, AppliedBiosystems) were used as endogenous control.

Cell Culture and Transfection.

HIT T15 cells and mouse islets were maintained in RPMI-1640 medium asdescribed previously (29). Labeling was undertaken with [3H]myo-inositol (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) 10or 50 μCi/ml for insulin-secreting HIT T15 cells and islets respectivelyin a special RPMI-1640 medium, described previously (29). Cells werelabeled for 72 h and labeling from 48-168 h did not change the InsP6 toInsP7 ratio. For experiments, islets or cells were transferred withwashing into a Krebs buffer and incubated for 30 min under basal glucoseconditions (0.1 mM for cell lines and 3 mM for islets), Inositolpolyphosphates were extracted and separated on HPLC as describedpreviously (29). INS-1E cells were cultured as described elsewhere (30).Mouse pancreatic islets were isolated from female NMRI mice(Bomholtgaard, Ry, Denmark) or normo-glycemic ob/ob mice as previouslydescribed (31,32). Cells were incubated in RPMI 1640 medium (InvitrogenCorporation, Carlsbad, Calif.) supplemented with 10% (v/v)heat-inactivated fetal calf serum, 100 IU/ml penicillin and 100 μg/mlstreptomycin. Single mouse islet cells were transfected adherently theday after plating with pIRES2-EGFP (mock) or a combination ofpIRES2-EGFP and construct of interest at 2 μg/ml in the above RPMI 1640cell culture medium using Lipofectamine™ 2000 (Invitrogen Corporation,Carlsbad, Calif.) according to manufacture's instructions,Lipofectamine™ was used in a ratio of 4:1 to DNA. Cells were used 48 hafter transfection. Based on GFP fluorescence, the transfectionefficiency in mouse islet cells amounted to 8+/−1% (n=124 cells; 4different cell preparations and transfections). SiRNA's were transfectedinto MIN6m9 cells and primary islet cells using Lipofectamine™ 2000 andOpti.MEM™ media. The medium was changed the following day into normalculture media for either MIN6m9 cells or (primary islet cells and thecells cultured for a further 4 days.

Capacitance Measurements.

Cells expressing EGFP were selected for capacitance measurements.Exocytosis was monitored as changes in cell capacitance using either theperforated patch or standard whole-cell configuration of the patch-clamptechnique and an EPC9 patch-clamp amplifier (Heka Elektronik,Lambrecht/Pfalz, Germany). The pipette solution for the perforated patchconfiguration consisted of (in mM) 76 Cs2SO4, 10 NaCl, 10 KCl, 1 MgCl2,5 HEPES (pH 7.35 with CsOH) and 0.24 mg/ml amphotericin B. Perforationrequired a few minutes, and the voltage clamp was consideredsatisfactory when the Gseries (series conductance) was stable and >35nS. The pipette solution used for standard whole-cell recordingscontained (in mM) 125 Cs-glutamate, 10 CsCl, 10 NaCl, 1 MgCl2, 5 HEPES,0.05 EGTA, 0.01 GTP and 3 MgATP (pH 7.15 using CsOH). InsP7 isomers weredissolved in the pipette-filling solution to the final concentrationsindicated in the text and kept on ice until use. The extracellularmedium was composed of (in mM) 118 NaCl, 20 tetraethylammonium-Cl, 5.6KCl, 1.2 MgCl2, 2.6 CaCl2, 5 HEPES (pH 7.40 using NaOH) and 3 glucose.The stimulation protocol consisted of trains of four 500-msdepolarizations applied at 1 Hz and went from −70 mV to zero mV. Thecapacitance measurements were performed at 33° C. and the recordingchamber was perfused at a rate of 1.5 ml/min.

Measurement of Single L-Type Ca2+ Channel Activity

Cell-attached patch recordings were performed in control MIN6m9 cellsand those subjected to IP6K1-siRNA as described previously (32).Briefly, typical electrode resistance was 2-4 MΩ Cell-attachedsingle-channel recordings were made with Ba2+ as the charge carrier (inmM): 110 BaCl2, 10 TEA-Cl, 5 HEPES-Ba(OH)2 and pH 7.4 and a depolarizingexternal recording solution, containing (in mM) 125 KCl, 30 KOH, 10EGTA, 2 CaCl2, 1 MgCl2, 5 HEPES-KOH and pH 7.15, is used to bring theintracellular potential to ˜0 mV. Recordings are made with an Axopatch™200 amplifier (Axon Instruments, Foster City, Calif.). Voltage pulses(200 ms) are applied at a frequency of 0.5 Hz to depolarize cells from aholding potential of −70 in V to a membrane potential of 0 mV. Resultingcurrents are filtered at 1 kHz, digitized at 5 kHz and analyzed with thesoftware program pCLAMP™ 6 (Axon Instruments, Foster City, Calif.,U.S.A.).

Human Growth Hormone (hGH) Release Assay.

After transfection with pCMV5-hGH and either empty vector pcDNA3 orplasmid of interest, INS-1E cells were seeded into 48-multiwell plates(2×10⁵ cells per well) and cultured for 48 h. Incubation and secretionexperiments were performed as described (33) using the sameextracellular medium as described above and supplemented with 3 mMglucose. hGH levels in the various samples were measured using ELISA(Roche, Mannheim, Germany).

Statistical analysis. Results are presented as mean values±S.E.M. forindicated number of experiments. Statistical significances wereevaluated using Dunnett's test for multiple comparisons to a control andTukey's test when multiple comparisons between groups were required.

Results

Using [3H] myo-inositol labelling protocols we examinedinsulin-secreting cells and pancreatic islets for the presence ofinositol pyrophosphate species. InsP7 was identified by its co-elutionwith a bone fide InsP7 standard generated using InsP6 kinase (data notshown). Very little InsP8 was detectable. FIG. 1A shows InsP7 levelsexpressed as a percentage of cellular InsP6 levels for aninsulin-secreting cell line or primary β-cells. In normal mousepancreatic islets (60% β-cels), the relative level of InsP7 is about 5%of the InsP6 level. In contrast, the percentage of InsP7 islets fromob/ob mice, which have more than about 90% β-cells, is about 8%. Thissuggests that the elevated levels are restricted to the β-cells.Normalizing the primary mouse data to 100% β-cells (FIG. 1B) suggeststhat they maintain InsP7 levels at about 9% of the InsP6 concentration.Of the insulin secreting cell lines, only HIT-T15 cells have a similarlevel of InsP7 (10% of InsP6). Using the equilibrium labellingtechniques (13) which can only be reliably applied to growing, culturedcells, we were able to estimate the basal concentration of InsP7 HIT-T15cells to be 5.8+/−0.14 μM, (±SEM, n=3), reflecting a concentration atthe top end of the range that has been estimated in other mammaliancells or yeast (1-5 μM) (4). Since InsP7 is in a state of rapid exchangewith the cellular InsP6 pool in β-cells (data not shown) in common withother mammalian cells (4) and the cellular concentration of InsP6 inβ-cells is also high (13), it is perhaps not surprising that high levelsof InsP7 exist in these cells.

An important caveat is that the high InsP7 is a cell-wide average whichdoesn't take into account separate cellular compartments. This isparticularly important as one of the main isoforms of InsP6 kinase,IP6K2, can be nuclear (9) and thus the InsP7 it produces may notinfluence events in the cytosol or plasma membrane, for example vesicletrafficking or exocytosis, respectively. Therefore, using Taqman™-basedquantitative Real time PCR we examined islet and β-cell lysates for thepresence of IP6K isoforms. FIG. 1C demonstrates the expression of IP6K1and IP6K2, but not IP6K3. Expression levels for the two kinases weresimilar in a given cell type, however, the expression of IP6K1 and 2 waslower in the primary cells compared to the cell line MIN6, perhapsreflecting the fact that InsP7 metabolism is up-regulated during thecell cycle (10,11). Thus the high InsP7 levels are not likely to reflectan exclusive nuclear pool but are likely to be consistently highthroughout the cell and thus could influence insulin secretion.

To investigate whether high InsP7 concentrations are responsible forkeeping β-cells in a responsive state, we over-expressed all 3 reportedmammalian IP6K's primary β-cells under basal conditions and examinedwhether stimulated exocytosis was subsequently enhanced. We usedincreases in cell capacitance as a measure of exocytosis. This techniquedetects the increase in β-cell surface area that occurs when theinsulin-containing granules fuse with the plasma membrane (17). Theperforated patch whole-cell technique was used to allow measurements inmetabolically intact cells and exocytosis was elicited by trainsconsistent of four 500-ms depolarizing pulses from −70 my to 0 mV. Inmock transfected cells, the capacitance increase elicited by the trainamounted to 79+/−11 fF (n=8; FIG. 2A, B). In cells overexpressing IP6K1the amplitude of the capacitance increase was stimulated by 153% andaveraged 198+/−12 fF (P<0.05; n=10), whereas no effect on exocytosis wasobserved in cells overexpressing a kinase-dead version of IP6K1 (FIG.2A, B). Interestingly, the capacitance increase evoked by the firstdepolarization was augmented by 293% in cells overexpressing wild-typeIP6K1. Exocytosis during the first depolarization is believed to largelyrepresent the content of the readily releasable pool (RRP)(18). The sizeof the RRP (in fF) can be estimated using the equation: RRP=S/(1−R2),where S is the sum of the response to the first (ΔC1) and the second(ΔC2) pulse and R is the ratio ΔC2/ΔC1 (18). We estimate that the RRPaveraged 96+/−9 fF (n=8) and 225+/−21 fF (n=10) in mock and wildtypeIP6K1 transfected cells, respectively. Thus, IP6K1 increased the size ofthe RRP by 134%. Using a conversion factor of 3 fF per granule (19), itcan be estimated that the RRP contains 30 and 75 granules in mock andwildtype IP6K1 transfected cells, respectively. The stimulatory actionof IP6K1 is restricted to the first depolarization and littleenhancement is seen during the final three pulses (FIG. 2B). Theexhaustion of the exocytotic response during the train is unlikely toreflect inactivation of the Ca2+ current with resulting suppression ofCa2f-induced exocytosis (FIG. 2C).

FIG. 2D shows that the ability of wild-type IP6K1 to stimulateexocytosis is shared by IP6K2 and IP6K3. Overexpression of a kinase-deadversion of IP6K2 and IP6K3 did not affect the exocytotic capacitycompared mock transfected cells (FIG. 2D). To confirm a role of IP6K'sin the control of exocytosis, we tested the effect of theiroverexpression in INS-1E cells using the hGH transient co-transfectionassay, in which hGH acts as a reporter of exocytosis from transfectedcells only. INS-1E cells represent a suitable cell system since totalincreases in cell capacitance in cells overexpressing IP6K1 werecomparable to those observed in primary mouse β cells (data not shown).Overexpression of IP6K1-3 stimulated hGH secretion 150% above basal(P<0.05; n=9-12), an effect that was not shared by their kinase-deadmutants. (FIG. 2E). Based on the fact that only IP6K1 and 2 are presentin β-cells, these and not IP6K3 are likely modulators of exocytosis.

An important concern is that IP6K's can also use InsP5 as a substrate,generating a different subset of inositol pyrophosphates (4). Therefore,it was necessary to verify that InsP7 is able to directly promoteexocytosis. The mammalian InsP7 is the 5-isomer and this was used indetailed experiments (FIG. 3A-D). We also assessed other theoreticalisomers of InsP7 (FIG. 3E). To measure the effects of 5-InsP7 onexocytosis, we applied trains of depolarizations in standard whole-cellexperiments where the β-cell was dialyzed with a solution containing 3μM InsP7. Following establishment of the whole-cell configuration, thecell was allowed two minutes equilibration period. A train consisting offour 500 ms depolarizations from −70 mV to 0 mV was then applied toevoke exocytosis. In a series of six experiments, the total increase incell capacitance amounted to 231+/−12 fF (P<0.01) in the presence of 3μM InsP7 in the pipette-filling solution and 77+/−11 fF under controlconditions, respectively (FIG. 3A). As was the case for cellsoverexpressing IP6K1-3 the capacitance increase evoked by the firstdepolarization in the presence of 5-InsP7 was strongly stimulated withonly little effect on exocytosis in response to the subsequent 3depolarizations (FIG. 3B). The ability of 5-InsP7 to stimulateexocytosis was not associated with a change in the whole-cell Ca2+current (FIG. 3C). The stimulatory action of 5-InsP7 exocytosis wasconcentration dependent (FIG. 3D). No stimulation of exocytosis wasobserved at ≦0.1 μM InsP7. At higher concentrations, 5-InsP7 stimulatedexocytosis by 90-410%. Approximating the average data points to theequation yielded a half-maximal stimulatory effect of 1.02 μM and aco-operativity factor of 1.5. Maximal stimulation of exocytosis wasobserved at concentrations of InsP7≧10 μM, which produced >380%stimulation (FIG. 4D). Thus, 5-InsP7 dose-dependently enhancesexocytosis within the physiological range of InsP7 concentrations (1-10μM). Other isomers of InsP7 were also able to stimulate exocytosis at 10μM, however CH-PP, a simple pyrophosphate based on cyclohexane, wasineffective (FIG. 4E). Under the conditions used to examine InsP7'seffect on exocytosis, the net effect of InsP6 was to promote endocytosisnot exocytosis (see FIG. 5). This is because the effect of InsP6 onexocytosis can only be discerned under conditions in which endocytosisis inhibited (15). This is not the case for InsP7. Furthermore, theeffect of InsP6 exocytosis, when endocytosis is inhibited, does notselectively promote secretion from the RRP (data not shown). Our dataillustrate that InsP7 and InsP6 have distinct effects on exocytosis.These experiments and those involved in overexpression of kinases do notpreclude a role for a more phosphorylated pyrophosphate i.e. InsP8,however since this pyrophosphate is either at a very low concentrationor undetectable in β-cells (data not shown), it is unlikely to play aphysiological role.

All our data to this point indicate a role for InsP7 in regulatedexocytosis, however our results are based on exogenous addition ofeither enzymes or InsP7. To test whether endogenous InsP7 contributes tothe exocytotic capacity in a physiologically relevant manner, wesilenced IP6K1 and IP6K2-cells using siRNA. Mouse-specific siRNA's werescreened using the mouse-cell line, MIN6 and Taqman™ Real time PCR geneexpression assays see FIG. 6). Elimination of either IP6K1 or IP6K2significantly reduced cellular InsP7 levels (see FIG. 7). Suitable siRNAcandidates were fluorescently tagged and transfected into primaryβ-cells. Cell capacitance measurements on fluorescent cells using theperforated patch technique described above were carried out.Interestingly, only the silencing of IP6K1 but not IP6K2 (FIG. 4C)inhibited the exocytotic capacity, and the effect of silencing was againmost pronounced on the first pulse reflecting depletion of the RRP ofgranules (FIGS. 4A,B). Furthermore, addition of 5-Ins/n7 in the wholecell mode when the IP6K1 had been silenced was able to restore normalexocytotic response (FIG. 4D). Thus endogenous InsP7 generated by IP6K1but not IP6K2 is responsible for the enhanced exocytotic capacity inpancreatic-cells. The discrepancy between our exogenous vs. endogenoussystems may reflect a differential distribution or cellular associationsof the 2 kinases in vivo. Indeed IP6K1 can associate with proteinsinvolved in exocytosis which IP6K2 cannot (20). Interestingly, otherstudies looking at the role of IP6K2 in apoptosis indicate a similarpattern (21). That is, substantial overexpression of IP6K1-3 leads to anincrease in apoptosis, however only the silencing of IP6K2 prevents it.In both cases the supra physiological increase of InsP7 clearlyovercomes some compartmentalization exhibited by the different kinases.

One possible mechanistic explanation for the effect of 5-InsP7exocytosis may be direct stimulation of voltage-gated L-type Ca2+channel activity, as previously shown for InsP6 (13). Although thewhole-cell Ca2+ channel data speak against this (FIGS. 2C and 3C), adetailed analysis was made applying the cell-attached patchconfiguration, maintaining an intact intracellular milieu, in MIN6m9cells subjected to IP6K1-siRNA, which significantly decreasesintracellular InsP7 (FIG. 7). As shown in FIG. 8, IP6K1 siRNA did notsignificantly alter channel number per patch, open probability, meanclosed time and mean open time (P>0.05). Hence, InsP7 does not affectL-type Ca2+ channel activity, which in striking contrast to InsP6 (13).

In summary, the pancreatic β-cell maintains high levels of InsP7. Thispyrophosphate then serves as an essential player in the insulinsecretory process by regulating the readily releasable pool ofinsulin-containing granules and thereby maintaining the immediateexocytotic capacity of the β-cell. An important question for the futureis whether disruption of InsP7 metabolism plays any role in thepathogenesis of type 2 diabetes, a disease characterized by a secretorydefect in the pancreatic β-cell (22). In this respect, hints areprovided by the putative disruption of the IP6K1 gene in a Japanesefamily with type 2 Diabetes (23) and the reduction of both plasmainsulin levels and glucose tolerance in mice in which the IP6K1 gene hasbeen deleted (24).

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1. A method for treating type II diabetes comprising administering to apatient with type II diabetes an effective amount of a therapeuticcapable of increasing expression of IP6K1 kinase.
 2. The method of claim1, wherein the increased expression of IP6K1 results in an increase inInsP₇ expression.
 3. A method for stimulating insulin exocytosis frompancreatic beta cells comprising administering to a patient in needthereof an effective amount of a therapeutic capable of increasingexpression IP6K1 kinase.
 4. The method of claim 3, wherein the increasedexpression of IP6K1 results in an increase in InsP₇ expression.
 5. Themethod of claim 1 wherein the therapeutic comprises a gene therapyvector capable of increasing the expression of IP6K1.
 6. The method ofclaim 1 wherein the therapeutic comprises IP6K1, or an active fragmentthereof.
 7. A method for treating type ii diabetes comprisingadministering to a patient with type II diabetes an effective amount ofa therapeutic capable of increasing production of InsP₇.
 8. A method foridentifying a compound for treating type II diabetes comprising: (a)contacting pancreatic beta cells with one or more test compounds; and(b) determining expression level of IP6K1 kinase and/or levels of InsP7;wherein an increase in the expression of IP6K1 kinase and/or an increasein InsP7 indicates that the compound is suitable for treating type IIdiabetes.